The invention relates to a method for diagnosis and/or prognosis of cancers, for diagnosis of the site of origin of tumor cells, for optimizing cancer therapy for patients and for screening active substances in oncology in which the mechanical properties of tumor cells and reference cells from normal tissue are analyzed and the diagnosis and/or prognosis of cancers is determined from the engineering strain of the cells resulting from the input of a directed mechanical stress. The invention is used in research, medicine and pharmacy.
The term cancer and cancers summarize an entire class of diseases, which have in common that they form malignant tumors. To date, more than 200 different tumors were identified. Characteristic of all malignant tumors is the uncontrolled proliferation of the cells which are able to displace healthy tissues (invasion of adjacent tissues), and can develop metastases in tissues of the entire body (distant metastases). These three processes are characteristic of the progression of cancer, and they are used as criteria for the classification of cancer in the cancer stages I, II, III and IV (or A-D, respectively) or for staging by means of the TNM-classification and thereby for making a statement regarding the aggressiveness of the disease. It can be determined from stage III (C) that the tumor grows beyond its formation region, and displaces the surrounding tissue. From Stage IV (D), distant metastases can be detected. Depending on the stage of cancer, different treatment is recommended in order to achieve successful recovery. In this respect, the assessment of the aggressiveness of tumor cells is of great importance to select the appropriate therapy method for a patient. Further, it is important that a classification can be made as early as possible in order to select an appropriate therapy. Until now, it is not possible, to determine based on primary tumor samples, whether an advanced stage with the formation of metastases is already present. Diagnosis of cancer is confirmed by the patient's symptoms using imaging methods, such as MRT, CT and the like, as well as by the detection of specific tumor markers. Tumor markers are predominantly proteins and peptides, which are detected in the blood or other body fluids of the patient or on the cell surface, their elevated concentration being indicative of a tumor. Since malignant tumor cells develop from mutated cells of normal tissue, the tumor markers are detectable in cells of normal tissue and are in tumors characterized only by their different frequency of occurrence. A plurality of different tumor markers have been associated with various cancers. Due to their low specificity for tumor cells, they are predominantly unsuitable for diagnostic purposes. Classification into cancer stages by means of tumor markers is currently not possible. For a final diagnosis, pathological examination of tumor sections is currently performed. To analyze how far a tumor has progressed, it is with current testing methods necessary to directly search for the tumor metastasis and to remove, for example suspicious tissue (e.g., lymph nodes).
In this manner and by using imaging methods, it is possible to classify the cancer. Metastasis can be determined only at the time when they have formed sufficiently large cell structures, so that they are visible with the respective method. An incipient metastasis, however, is difficult to prove.
Differing from the widespread analysis of proteins, in particular surface proteins, as tumor markers, and thereby from the analysis of biochemical properties of the cell for diagnosis or prognosis of cancer, it has been found that, compared with cells from normal tissue, tumor cells have undergone changes in the cytoskeleton resulting in a change in the mechanical (herein also “biomechanical”) properties of the cell.
It is known that based on the mechanical properties of the cytoskeleton of cells, a statement can be made about the proliferation of the cells [Lekka at al. 1999, Guck et al. 2005, Cross et al. 2007, Remmerbach et al. 2009]. It was found that tumor cells, i.e. cells which proliferate uncontrollably, have a higher deformability compared with normal tissue. Further increased deformation was for breast cancer also associated with a risk for the migration of cells and thus the formation of metastases [Guck et al. 2005, Ward et al. 1991]. This higher deformability of the tumor cells was accounted for by the fact that actin filaments of the cells being present in the interphase are regressed with the cell entering into mitosis and actin is thereafter present in diffuse distribution in the cytoplasm of cells [Sanger et al. 1975]. It is further assumed that actin is downregulated in tumor cells [Rao & Cohen 1991]. Especially actin in the form of filaments aids stabilizing the cells, so that higher deformability of the cells can be used as a suitable marker for the uncontrolled proliferation of cells. This increased deformability of the tumor cells is observed under mechanical loads, which result in linear deformation of the cell. Besides actin, other cytoskeleton elements such as microtubules, intermediate filaments and their associated crosslink and motor proteins have influence on cell mechanics.
U.S. Pat. No. 6,067,859 with the so-called optical stretcher discloses a method with which deformation of cells can be caused under a load. In this, deforming mechanical stress is by means of laser beams in opposite direction applied to the cells to be analyzed resulting in viscoelastic deformation of the cell. The tension stress being generated by the laser beams at the time of load application (application of the mechanical stress) results in an elongation of the cell along the major axis of the cell, which is oriented along the laser beams. The engineering strain thus determined, i.e. the relative change in length of the cell along the direction of direction of stressing is greater for tumor cells than for normal tissue. The change in length of the cell is optically monitored by a microscope, so that the engineering strain can be determined.
Alone the statement that cells have higher deformability is not yet sufficient for any diagnosis or prognosis of cancer. Also other cells currently being in the midst of division exhibit such changes of the cytoskeleton. To establish a reliable prognosis of cancer, a statement about the likelihood of metastases or the invasion of the cells into surrounding tissue, respectively, must be made. As already stated, metastasis can currently only be determined at a relatively late stage, namely after the formation of metastases, by means of imaging methods.
There is therefore a need to characterize metastatic or invasive cells by specific properties of the cytoskeleton and use these properties for the diagnosis and/or prognosis of cancers. By means of special biomechanical properties being associated with the likelihood of metastases or invasiveness of cells, there is a new therapeutic approach in cancer therapy, in that the mechanical properties of the cell are changed. There is also a need for thus selecting active substances for the treatment of a patient by patient-specific active substance screening, so as to improve the chances of recovery.
The object of the invention is to provide a method by means of which a statement regarding the risk of tumor metastases and possibly the presence of invasive cells in a patient can be made by analyzing previously unknown mechanical properties of tumor cells. It is a further object of the invention to provide a screening method used to identify the potential active substances for cancer therapy which affect the biomechanical properties of the tumor cells. Another object of the invention is to enable an individualized therapy adapted to the needs of the patient by specific active substance screening. Furthermore, it is an object of the invention to draw conclusions about the site of origin of a tumor by means of analyzing the biomechanical properties of tumor cells of a patient.